Localization of antigenic sites on Der p 2 using oligonucleotide-directed mutagenesis targeted to predicted surface residues.

Abstract

Understanding the molecular nature of allergen-antibody interactions is important to understanding the mechanism of conventional immunotherapy as well as to designing alternative immunotherapeutic strategies. Many important allergens have been cloned and expressed, making it possible to apply recombinant DNA techniques to dissect antigenic determinants. The aim of this study was to use predictive algorithms and site-directed mutagenesis to investigate monoclonal antibody and IgE antibody epitopes of the major house dust mite allergen Der p 2. Computer algorithms were used to assess the primary amino acid sequence of Der p 2 and to identify regions of hydrophilic and flexible sequence. Subsequently, site-directed mutagenesis was used to generate amino acid substitutions at hydrophilic residues at positions 44-46 and at position 100. The variants were tested in a competitive inhibition ELISA with four group 2-specific murine monoclonal antibodies and with human IgE antibody from mite allergic patients. Conservative amino acid substitutions at position 44-46 did not distinguish IgE antibody epitopes, but did suggest that these residues are involved in the epitope defined by one monoclonal antibody, 15E11. Non-conservative substitution of proline at this position reduced binding to all four monoclonal antibodies, as well as IgE antibody, by 50-80%. Point mutants at position 100 mapped the epitopes of two monoclonal antibodies, 7A1 and 13A4, previously shown to bind the same region of Der p 2. In addition, the two variants tested at this position showed distinct inhibition curves with these two monoclonal antibodies indicating differences in fine specificity. Using predictive algorithms, in the absence of tertiary structural information, we have been able to localize important B cell determinants on Der p 2. The results suggest that it is possible to modulate antibody recognition of allergens using site-directed mutagenesis and that this approach may provide a new strategy for allergen specific immunotherapy.